Nanoelectromechanical system (NEMS) resonators are electronically and optically controllable, submicron-scale mechanical cantilevers that can be used for exceptionally sensitive mass detection of analytes. Upon adsorption onto a NEMS resonator, analytes can precipitously downshift a resonant frequency of the resonator, which is continuously monitored by specialized electronic circuitry. The induced frequency change is proportional to the mass of the molecule and depends on the landing position on the resonator. Technical solutions enabling this technology can be found, for example, in U.S. Pat. Nos. 6,722,200; 7,302,856; 7,330,795; 7,552,645; 7,555,938; 7,617,736; 7,724,103; 8,044,556; 8,329,452, 8,350,578; and 9,016,125.
These developments have been applied to ultra-sensitive mass detection of biomolecules, including single molecules, as described in, for example, U.S. Pat. Nos. 6,722,200; 8,227,747 and US Patent Publication 2014/156,224. Simple spectra have been assembled by statistical analysis from only a few hundred molecular adsorption events, and in the latest embodiments, with individual molecules.
One of problems needed to be solved for single-molecule analysis was that the resonant frequency shift induced by analyte adsorption depends upon both the mass of the analyte and its precise location of adsorption upon the NEMS resonator. This problem was solved by exciting and detecting multiple vibration modes of the resonator to determine both of these unknowns, as described in US Patent Publication 2014/156,224. Mass resolution of 50-100 kDa has been demonstrated, with significant improvements expected as technology develops further. See, for example, Yang, Y. T., et al., Zeptogram-scale nanomechanical mass sensing. Nano Letters 6, 583-586 (2006).
Nanospray ion source and MS atmosphere-to-vacuum interface were used together with NEMS detection in US Patent Publication 2014/156,224, as well as matrix-assisted laser desorption and ionization source. Cooling the NEMS enhanced non-specific physical adsorption of the arriving analytes on the surface of the devices. By individually measuring the mass of sequentially arriving analyte particles, a mass spectrum representing an entire heterogeneous sample was constructed in US Patent Publication 2014/156,224.
By continuous monitoring of multiple vibrational modes, this approach was then extended in US Patent Publication 2014/244,180 to detection of spatial moments of mass distribution for individual analyte entities, one-by-one, as they adsorb onto a nanomechanical resonator.
Hence, NEMS has become a viable approach to mass spectrometry (MS, NEMS-MS). Particularly important is that NEMS-MS can be used to evaluate neutral molecular species and also that resolving power and sensitivity improves with increasing mass.
Mass spectrometry traditionally addresses identification of analytes by first supplying them with charge in an ion source and then measuring analyte mass-to-charge ratio using electromagnetic fields. In recent years, mass spectrometry has assumed an increasingly important role in the life sciences and medicine and became the main technology for proteomic analysis. Increasing resolution and mass range of modern mass analyzers allows one to measure protein complexes and even virus capsids up to 1-50 MDa using nanospray at native conditions (i.e., at pH close to physiological). For example, it was shown in U.S. Pat. No. 8,791,409 that orbital electrostatic trap mass spectrometry could detect individual ions of protein complexes with mass resolving power in thousands. However, MS based on mass-to-charge ratio typically shows decreasing performance at higher masses, especially because of overlapping charge distributions of MDa analytes.
Despite advances in the art, a need exists to continue to improve and make more versatile the capabilities of mass spectrometry, particularly for solving complex analytical problems with respect to large and complicated biological molecular structures, complexes, and even organelles found in the life sciences. Better methods are needed to determine the vast information available ranging from primary sequence determination to higher-order structure and dynamics for proteins and complexes. Charge state assignment can be difficult for larger, poorly desolvated protein complexes.